Vascular shear stress increases when blood flow or blood viscosity increases or when vessel diameter decreases. In the systemic circulation, shear stress is a potent stimulus for endothelial nitric oxide synthesis. We studied isolated rat lungs to determine whether increasing shear stress increases nitric oxide synthesis in the pulmonary circulation. Lungs were given the vasoconstrictor, U46619 (a thromboxane analogue), and perfused at constant flow rates or at constant pressure, since constant pressure perfusion minimizes changes in shear stress with vasoconstriction. The subsequent effect of the NOS inhibitor, N omega-methyl-L-arginine (LMA), or the soluble guanylyl cyclase inhibitor, 6-anilino-5,8-quinolinodione (LY83583) was assessed. Changes in pulmonary vascular resistance (PVR), pulmonary vascular compliance, and perfusate cyclic GMP concentration were measured as indicators of nitric oxide synthesis. The effect of the cyclic GMP-specific (type V) phosphodiesterase inhibitor, zaprinast, on perfusate cyclic GMP concentrations was also examined. An infusion of U46619 consistently increased PVR and decreased compliance. LMA and LY83583 also increased PVR in U46619-treated lungs perfused at constant flow rates, primarily by increasing precapillary resistance. LMA had no effect in U46619-treated lungs perfused at constant pressure. Perfusate cyclic GMP concentrations increased significantly after U46619 in lungs perfused at constant flow rates, but cyclic GMP levels did not change after U46619 in lungs perfused at constant pressure. Zaprinast also increased perfusate cyclic GMP, demonstrating that increases in intracellular cyclic GMP are reflected in circulating cyclic GMP concentrations. We conclude that vasoconstriction with U46619 increases nitric oxide synthesis in isolated rat lungs. Lungs perfused at constant pressure respond differently to NOS inhibitors compared to those perfused at constant flow, suggesting that shear stress may increase nitric oxide synthesis in the lung. Perfusate concentrations of cyclic GMP reflect activation of soluble guanylyl cyclase in this model.